Viral vector-mediated gene delivery to the nervous system has proven a powerful methodology with both basic and therapeutic applications. Recombinant AAV vectors and lentiviral vectors, in particular, are increasingly popular in this context because of their simplicity, excellent safety profiles, and durable long-term gene expression. However, the transgene capacity of these vectors is limited to accommodate up to 4.5 and 8 kb of exogenous DNA, respectively. We have been studying and developing the HSV-based amplicon system as an alternative gene delivery system over the past decade. HSV amplicon vectors represent plasmid-based, replication-deficient vectors that carry less than 1% of the entire HSV genome and that include an origin of DNA replication and a DNA cleavage/packaging signal. The major strength of the vector system is its large transgene capacity to accommodate up to 150 kb of exogenous DNA. This unique feature allows amplicon vectors to incorporate and deliver an entire genomic locus, multiple expression cassettes, inducible systems for regulated gene expression, and reporter genes to enable quantitation and visualization of fusion proteins, promoter activity, or transduced cells. Development and use of such multi-functional amplicon vectors will undoubtedly impact research in both basic and translational neuroscience. For example, an indicator amplicon vector could be engineered that expresses one or multiple reporter genes for real-time monitoring of various cellular activities or a regulatable amplicon vector could be created that expresses one or multiple transgenes and/or short hairpin (sh) RNAs under the control of a cell type-specific or inducible promoter for regulated expression of exogenous and/or endogenous genes of interest. Significantly, such complex genetic constructs with sizes up to 150 kb can be readily transferred to virtually any HSV-infectable cells in culture and even in living animals. However, construction of such complex vector plasmids requires well designed cloning strategies and considerable technical skills. Moreover, exchanging one DNA element for another or adding a new DNA element to such complex vector plasmids often requires redesign of the entire construct and extensive molecular cloning procedures. To overcome these problems, we have come up with a new strategy in which DNA elements and expression cassettes are individually cloned into separate shuttle plasmids and then assembled into a single amplicon plasmid by sequential use of multiple site-specific DNA recombinases. In this proposed project, we will develop such an efficient and versatile tool kit to build complex HSV amplicon vector plasmids and demonstrate their usefulness in facilitating wider use and full exploitation of HSV amplicon vectors in neuroscience applications. PUBLIC HEALTH RELEVANCE: Viral vector delivery of genes to the nervous system has proven a powerful approach with both basic and therapeutic applications. We propose to develop and improve a herpes simplex virus (HSV)-based amplicon vector system for neuroscience research. The work may provide new methodologies to study the normal development of the nervous system and the pathogenesis of neurological disorders, and the developed technologies may be applied to improving novel therapeutics for neurological disorders.